Alanine transaminase enzymes and methods of use

ABSTRACT

Novel alanine transaminase (ALT) polypeptides and the use thereof as a diagnostic marker to predict and monitor tissue damage and/or tissue malfunction. The ALT polypeptides are murine and/or  rattus  ALT polypeptides and said ALT polypeptides are used to detect, predict and/or determine hepatic processes of an animal, particularly mice and/or rats.

GOVERNMENT INTEREST

This work was supported in part by grant R03 DK60563-01 from theNational Institutes of Health. The U.S. Government has certain rights tothis invention.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. provisional patentapplication Ser. No. 60/563,389, filed on 19 Apr. 2004, and U.S.provisional patent application Ser. No. 60/588,126, filed on 15 Jul.2004. The priority provisional patent applications are herebyincorporated by reference herein in their entirety and are made a parthereof, including but not limited to those portions which specificallyappear hereinafter.

FIELD OF THE INVENTION

This invention relates to alanine transaminase (ALT) polypeptides andthe use thereof as a diagnostic marker to predict and monitor tissuedamage and/or tissue malfunction. More specifically, the ALTpolypeptides are murine and/or rattus ALT polypeptides and said ALTpolypeptides are used to detect, predict and/or determine hepaticprocesses of an animal, particularly mice and/or rats. The presentinvention additionally relates to assays for the ALT polypeptides todiagnose tissue damage and/or tissue malfunction having a range ofetiologies that include but are not limited to hepatitis, nonalcoholicsteatohepatitis (NASH), fatty liver, cirrhosis, and drug hepatotoxicity,and other disorders in muscle, brain, kidney, and adipose tissue,particularly in mice and/or rats.

BACKGROUND OF THE INVENTION

Alanine transaminase (ALT) [EC 2.6.1.2., also known as glutamatepyruvate transaminase (GPT) and alanine aminotransferase] is a pyridoxalenzyme catalyzing reversible transamination between alanine and2-oxoglutarate to form pyruvate and glutamate. By mediating theconversion of these four major intermediate metabolites, ALT plays animportant role in gluconeogenesis and amino acid metabolism. In muscleand certain other tissues, ALT degrades amino acids for fuel, and aminogroups are collected from glutamate by transamination. ALT transfers theα-amino group from glutamate to pyruvate to form alanine, which is amajor amino acid in blood during fasting. Alanine is taken up by theliver for generating glucose from pyruvate in a reverse ALT reaction,constituting the so-called alanine-glucose cycle. This cycle is alsoimportant during intensive exercise when skeletal muscles operateanaerobically, producing not only ammonia groups from protein breakdownbut also large amounts of pyruvate from glycolysis.

ALT activities exist in many tissues, including liver, muscle, heart,kidney, and brain. Molecular cloning of the complementary DNAs (cDNAs)of two human ALT isoenzymes, hALT1 and hALT2 have been disclosed inInternational Publication WO 02/092768, herein fully incorporated byreference in its entirety. The independent DNA encoding for the twohuman ALT isoenzymes (gpt1 and gpt2, respectively) has been shown to belocalized to separate chromosomes in humans, and that they havedistinctive tissue distribution patterns, suggesting a tissue-dependentrole for ALT isoenzymes.

Perhaps the most well-known aspect of ALT is that it is used clinicallyas an index of liver integrity or hepatocellular damage. Serum ALTactivity is significantly elevated in a variety of liver damageconditions including viral infection, alcoholic steatosis, nonalcoholicsteatohepatitis (NASH), and drug toxicity, although the underlyingmechanism is generally not well understood. While low level of ALT ispresent in peripheral circulation because of normal cell turnover orrelease from nonvascular sources, the liver has been shown to containthe highest levels of ALT. The difference between ALT levels in liverand in blood has been shown to be about 2,000-3,000-fold. Hence, theincreased ALT in serum, plasma, or blood is regarded as a marker ofliver injury because of the “leakage” of hepatic ALT into thecirculation. Usually, the nature of liver injury causes the blood ALTlevels to vary greatly. Extremely high transaminase levels (greater than8- to 10-fold normal) can indicate acute viral hepatitis and/ordrug-induced hepatotoxicity. A mild chronic increase of serum ALT (2- to8-fold) is generally a characteristic of chronic hepatitis, fatty liver,and/or steatosis. However, many details of the mechanism for thecorrelation of ALT levels with the etiology of liver damage remain to beunderstood.

Even though serum ALT is one of the most widely-used assays in clinicalchemistry, there are serious deficiencies with the assay because it isan inadequate predictor in some cases. Recent studies have cast doubt onserum ALT assay's specificity for liver disease. Higher than normal ALTlevels are frequently associated with other clinical conditions such asobesity, muscle disease, heart failure, hemochromatosis, Wilson'sdisease, α1-antitrypsin deficiency.

There is a need for improved ALT immunoassays that more accuratelyindicate and/or diagnose tissue injury and/or disease. There is a needfor an ALT animal model for research and testing purposes. There is alsoa need for improved animal ALT immunoassays for use in, for example,drug testing and toxicology studies.

SUMMARY OF THE INVENTION

As discussed above, alanine aminotransferase (ALT) is a widely usedindex of liver integrity or hepatocellular damage in clinics as well asa key enzyme in intermediatary metabolism. Complementary DNAs of murinehomologues of human alanine aminotransferase 1 and 2 (mALT1 and mALT2)and of rat homologues of human alanine aminotransferase 1 and 2 (rALT1and rALT2) have been cloned.

The polypeptides of murine ALT1 (mALT1) and ALT2 (mALT2) of thisinvention share 87% and 93% identity, respectively, with their humancounterparts at the amino acid level. The murine ALT genes of the twomurine ALT isoenzymes localize to separate chromosomes, with the murineALT1 gene (gpt1) on chromosome 15 and the murine ALT2 gene (gpt2) onchromosome 8. The murine gpt1 and gpt2 also differ in messenger RNAexpression. The murine gpt1 is mainly expressed in liver, bowel, andwhite adipose tissue (WAT) and the murine gpt2 is highly expressed inmuscle, liver, and white adipose tissue. Expression of recombinantmurine ALT1 and murine ALT2 proteins in Escherichia coli (E. coli)produced functional enzymes that catalyze alanine transamination.

Rat ALT1 polypeptide consists of 496 amino acids and shares 97% and 88%identity to murine and human ALT1, respectively, at the amino acidlevel. Rat ALT2 polypeptide is composed of 522 amino acids and share 98%and 95% identity to murine and human ALT2, respectively, at the aminoacid level. Rat ALT1 and rat ALT2 polypeptides have 68% sequenceidentity and 77% similarity. The genes of rat ALT1 and ALT2 reside onthe chromosome 7 and 19, respectively. A sequence alignment of murineALT1 and ALT2, human ALT1 and ALT2 and rat ALT1 and ALT2 is provided inFIG. 6.

The diagnostic value of murine ALT isoenzymes in liver disease wasdetermined by an obese animal model. In fatty livers of obese mice,murine ALT2 gene expression is induced 2-fold, but murine ALT1 remainsthe same. Furthermore, in fatty liver, total hepatic murine ALT activityis elevated significantly by 30% whereas aspartate aminotransferase(AST) activity remains unchanged. Thus, murine ALT2 is responsible forthe increased ALT activity in hepatic steatosis and allows for a murineALT isoenzyme-specific assay having more diagnostic value than total ALTactivity assays currently in clinical use. As many pharmaceuticalcompanies do preclinical toxicology experiments in mice by measuring ALTas an indicator of liver toxicity, the murine isoenzyme-specific assaysof this invention provide improved assays for assessing preclinicaltoxicity of new medications.

Another embodiment of the present invention is directed to antibodies,particularly anti-ALT1 antibodies and anti-ALT2 antibodies. In aspecific embodiment, the antibody specifically binds to murine ALT1. Inanother specific embodiment, the antibody specifically binds to murineALT2. In yet another specific embodiment, the antibody specificallybinds to rat ALT1. In still yet another specific embodiment, theantibody specifically binds to rat ALT2.

It is an object of this invention to have a murine ALT polypeptide whichhas the amino acid sequence of SEQ ID NO:1 (murine ALT1) or an aminoacid having about 95% homology thereto. In certain specific embodiments,the amino acid having about 95% homology to SEQ ID NO:1 is SEQ ID NO:6.

It is another object of this invention to have a murine ALT polypeptidewhich has an amino acid of SEQ ID NO:2 (murine ALT2) or an amino acidhaving about 95% homology thereto. In certain specific embodiments, theamino acid having about 95% homology to SEQ ID NO:2 is SEQ ID NO:5.

It is another object of this invention to have a polynucleotide whichencodes for each of the murine ALT isoenzymes. It is a further object ofthis invention that the polynucleotide encodes the amino acid sequenceof SEQ ID NO:1 and/or SEQ ID NO:2 or an amino acid sequence having about95% homology to SEQ ID NO:1 and/or SEQ ID NO:2.

It is also further object of this invention that the polynucleotidesequence be the sequence of SEQ ID NO:3 or SEQ ID NO:4. In oneembodiment in which the polynucleotide sequence is the polynucleotidesequence encoding for the homolog of SEQ ID NO:1 or SEQ ID NO:2, thepolynucleotide sequences are SEQ ID NO:7 (rat ALT1) or SEQ ID NO:8 (ratALT2).

It is another object of this invention to have a polynucleotide whichencodes for each of the rat ALT isoenzymes. It is a further object ofthis invention that the polynucleotide encodes the amino acid sequenceof SEQ ID NO:5 and/or SEQ ID NO:6 or an amino acid sequence having about95% homology to SEQ ID NO:5 and/or SEQ ID NO:6. It is a further objectof this invention that the polynucleotide sequence be the sequence ofSEQ ID NO:7 or SEQ ID NO:8.

It is another object of this invention to have an antibody which bindsspecifically to one of the isoenzymes of ALT and not the otherisoenzyme. For example, an antibody of one embodiment of this inventionis specific for murine ALT2 and does not bind to murine ALT1. In analternative embodiment, an antibody of the present invention is specificfor rat ALT2 polypeptide and not rat ALT1 polypeptide. In embodimentswhere a mouse animal model is employed, the antibody of the presentinvention binds to the murine ALT2 sequence of SEQ ID NO:2 or anALT2-specific fragment thereof or a homolog of SEQ ID NO:2, or,alternatively, to the protein encoded by the DNA sequence of SEQ ID NO:4or a murine ALT2-specific fragment thereof.

In an alternative embodiment, such as for use in a rat animal model, anantibody of the present invention specifically binds a rat ALT2 aminoacid sequence of SEQ ID NO:5 or a fragment or homolog thereof. Inanother embodiment, the antibody of the present invention specificallybinds a rat ALT1 amino acid sequence of SEQ ID NO:6 or a fragment orhomolog thereof.

It is an object of this invention to have an expression vector for eachof the ALT isoenzymes. The expression vector can be a plasmid, cosmid,or other type of vector where the DNA sequence encoding for the ALT isoperatively linked to expression sequences, such as a promoter. The DNAsequence for murine ALT can be the sequence of SEQ ID NO:3 and/or SEQ IDNO:4, or can be a sequence which encodes for the amino acid sequence ofSEQ ID NO:1 and/or SEQ ID NO:2 or a homolog of SEQ ID NO:1 and/or SEQ IDNO:2. The DNA sequence for rat ALT can be the sequence of SEQ ID NO:7(rALT1) and/or SEQ ID NO:8 (rALT2), or can be a sequence which encodesfor the amino acid sequence of SEQ ID NO:5 and/or SEQ ID NO:6 or ahomolog of SEQ ID NO:5 and/or SEQ ID NO:6.

It is an object of this invention to have a method for detecting thepresence of ALT1 mRNA and/or ALT2 mRNA in a sample. It is a furtherobject of this invention that the sample can be tissue or bodily fluidsfrom a mouse and/or rat. It is a further object of this invention that apolynucleotide probe be used to detect the presence of the ALT1 mRNAand/or the ALT2 mRNA in a sample.

It is an object of this invention to have a method to detect thepresence of ALT1 protein and/or ALT2 protein in a sample. It is afurther object of this invention that the sample can be tissue or bodilyfluids from a mouse and/or a rat. It is another object of this inventionthat one uses antibodies (monoclonal or polyclonal) that bindspecifically to ALT1 or that bind specifically to ALT2 to detect therespective protein. It is another object of this invention that thebodily fluids can be blood, serum, lymph, urine, sweat, mucus, sputum,saliva, semen, spinal fluid, interstitial fluid, synovial fluid,cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.It is also an object of this invention that the tissue can be liver,brain, muscle, adipose tissue, and kidney.

It is another object of this invention to have a method for diagnosingor detecting injury or disease involving tissue which contains ALT2. Itis a further object of this invention that the method involves usingantibodies (polyclonal or monoclonal) that specifically bind to a ALT2polypeptide of the present invention to measure the level of ALT2 inbodily fluids from the animal. It is another object of this invention touse antibodies (polyclonal or monoclonal) that specifically bind to ALT1to measure the level of ALT1 in bodily fluids from the animal and thento compare the level of ALT2 to ALT1. When the level of ALT2 issufficiently higher than the level of ALT1 or the level of ALT2 fallswithin a pre-determined range, then the animal is diagnosed with aspecific disease or injury. It is another object of this invention thatthe bodily fluids can be blood, serum, lymph, urine, sweat, mucus,sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid,cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.Furthermore, the tissue can be liver, brain, muscle, adipose tissue(white adipose tissue “WAT” or brown adipose tissue “BAT”), and kidney.

It is an object of this invention to have a kit useful in diagnosingdamage or disease in tissue containing ALT. This kit has a measurer ofALT, either ALT1 or ALT2, levels in a sample of bodily fluids and anindicator for determining if amount of ALT measured by the ALT measurerfalls in a range associated with damage or a specific disease in the ALTcontaining tissue. It is further object of this invention that the kitmay also contain a measurer for both ALT1 and ALT2 levels in a sample ofbodily fluids and an indicator for determining if amount of each of ALT1and ALT2 measured by the measurer(s) falls in a range associated withdamage or a specific disease in the ALT containing tissue. The ALT1measurer and the ALT2 measurer can be a biologic assay, anantibody-based assay, an enzyme linked immunosorbent assay, a Westernblot, a rapid immunoassay, a radioimmunoassay, and combinations thereof.

It is another object of this invention to have a diagnostic kit usefulfor diagnosing damage or disease to ALT1 containing tissue and/or ALT2containing tissue. This diagnostic kit can contain ALT1 specificantibodies (polyclonal or monoclonal), immunoassay reagents, and apositive and negative control. This kit can also have ALT2 specificantibodies (polyclonal or monoclonal). This kit includes a means fordetermining if a measurement of ALT1 and/or ALT2 indicates a diagnosisof damage or disease in ALT1 containing tissue and/or ALT2 containingtissue. The kit can also have instructions indicating when a level ofALT1 and/or ALT2 is indicative for diagnosis of damage or disease intissue containing ALT1 or ALT2.

It is still another object of this invention to have a kit useful indetermining when there are altered levels of ALT2 in bodily fluids(altered can be higher than normal or lower than normal). This kit canhave a measurer of ALT2 levels in a bodily fluids sample and anindicator for determining if the ALT2 level measured falls in a rangeassociated with a specific condition. It is a further object of thisinvention that the kit can determine when there are altered levels ofALT1 in bodily fluids (altered can be higher than normal or lower thannormal). This kit can also have a measurer of ALT1 levels in a bodilyfluids sample and another indicator for determining if the ALT1 levelmeasured falls in a range associated with a specific condition.Furthermore, this kit can have a third indicator for comparing thevalues of ALT1 and ALT2 and determining if the levels of ALT1 and ALT2fall in a range associated with a specific condition. The measurer ofthis kit can be selected from one or more of the following: a biologicassay, an antibody-based assay, an enzyme linked immunosorbent assay, aWestern blot, a rapid immunoassay, a radioimmunoassay, and combinationsthereof.

It is an object of this invention to have a method for producing a ALTpolypeptide of the present invention. The ALT produced can be the sameas the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:5or SEQ ID NO:6, or a homolog, fragment, or variant thereof. This methodinvolves cloning the DNA encoding for ALT in an expression vector,introducing the expression vector into a host cell to produce arecombinant host cell, and subjecting to the recombinant host cell toconditions such that ALT is expressed. It is a further object of thisinvention that the ALT expressed can be isolated and purified. The DNAsequence placed in the plasmid can be the nucleic acid sequence of SEQID NO:3 or SEQ ID NO:4 or SEQ ID NO:7 or SEQ ID NO:8, or a nucleic acidsequence which encodes for a variant, homolog, or fragment of murineALT1 or murine ALT2. Alternatively, the DNA sequence inserted into theplasmid may be a nucleic acid sequence which hybridizes, such as, forexample, under conditions of high stringency, to a nucleic acid encodinga polypeptide of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:5 or SEQ IDNO:6. In a specific embodiment, the high stringency conditions include0.1×SSC with 0.1% SDS wash buffer at hybridization temperature, such as,for example, at about 60, 61, 62, 63, 64, 65, 66, 67, or 68 degrees C.

It is another object of this invention to have a method for diagnosing acondition associated by altered levels of ALT2 and/or ALT1 in bodilyfluids in an animal, particularly a mouse and/or a rat. This methodinvolves contacting a sample of bodily fluids with at least one antibodywhich specifically binds to an ALT2 of the present invention, detectingthe ALT2 antibody which is bound to ALT2, and comparing the amount ofdetected ALT2 antibody to a known quantity for an animal without thecondition. In this method when the quantity of detected ALT2 antibodydiffers sufficiently from the known quantity from an animal without thecondition, then it indicates that the animal has the condition. Inaddition, the method also can involve contacting the sample of bodilyfluids with at least one antibody which specifically binds to ALT1,detecting the ALT1 antibody which is bound to ALT1, and comparing saidamount of detected ALT 1 antibody to a known quantity for an animalwithout the condition. In this method, when the quantity of detectedALT1 antibody differs sufficiently from the known quantity from ananimal without the condition, then it indicates that the animal has thecondition. Furthermore, this method can also involve comparing theamount of ALT2 antibody detected to the total amount of antibodydetected and/or to the amount of ALT1 antibody detect; and/or the amountof ALT1 antibody detected to the total amount of antibody detectedand/or to the amount of ALT2 antibody detected. Again, the condition isindicated if the amount of ALT2 antibody detected when compared to theamount of ALT 1 antibody detected or the total amount of antibodydetected falls within a certain range.

Again, the condition is indicated if the amount of ALT1 antibodydetected when compared to the amount of ALT2 antibody detected or thetotal amount of antibody detected falls within a certain range. It is afurther object of this invention that the bodily fluids for this methodcan be selected from the following group: blood, serum, lymph, urine,sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid,synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, andpleural fluid.

One embodiment of the invention is an isolated and purified murine ALTpolypeptide comprising the amino acid sequence selected from a groupincluding SEQ ID NO:1 and SEQ ID NO: 2. Another embodiment of theinvention is an isolated and purified polynucleotide encoding for themurine ALT polypeptide. The isolated and purified polynucleotide of oneembodiment comprises a polynucleotide sequence selected from a groupincluding SEQ ID NO:3 and SEQ ID NO:4. The invention also includes anisolated and purified antibody which binds specifically to the murineALT polypeptides of the present invention. The invention furtherincludes an expression vector for murine ALT comprising the murine ALTpolynucleotide sequence operatively linked to an expression sequence.

Another embodiment of the invention is an isolated and purified rat ALTpolypeptide comprising the amino acid sequence selected from a groupincluding SEQ ID NO:5 and SEQ ID NO: 6. Another embodiment of theinvention is an isolated and purified polynucleotide encoding for therat ALT polypeptide. The isolated and purified polynucleotide of oneembodiment comprises a polynucleotide sequence of SEQ ID NO:7 or SEQ IDNO:8. The invention also includes an isolated and purified antibodywhich binds specifically to the rat ALT polypeptides of the presentinvention. The invention further includes an expression vector for ratALT comprising the rat ALT polynucleotide sequence operatively linked toan expression sequence.

Another embodiment of the invention is a method of detecting in a samplethe presence of mRNA, wherein the mRNA encodes for an ALT polypeptide ofthe present invention. The method comprises contacting the sample with apolynucleotide probe, wherein the polynucleotide probe is sufficient tospecifically detect under stringent hybridization conditions thepresence of the mRNA, and detecting the formation of a hybrid of thepolynucleotide probe and the mRNA.

Another embodiment of the invention is a method of detecting an ALTpolypeptide of the present invention in a sample, wherein the samplecomprises a bodily fluid, the method comprising contacting a sample ofthe bodily fluids with at least one antibody that specifically binds tothe ALT polypeptide and detecting the antibody which is bound to the ALTpolypeptide in the sample.

Yet another embodiment of the invention is a method of diagnosing ordetecting injury or disease involving tissue which contains an ALTpolypeptide of the present invention in an animal suspected of havingthe injury or disease. The method comprises: contacting a sample ofbodily fluids from the animal with at least one first antibody, whereinthe first antibody specifically binds to an ALT1 polypeptide; detectingthe first antibody which is bound to the ALT1 polypeptide in the sample;contacting the sample of bodily fluids with at least one second antibodywherein the second antibody specifically binds to an ALT2 polypeptide;detecting the second antibody which is bound to the ALT2 polypeptide inthe sample; and comparing the amount of the ALT1 polypeptide bound tothe first antibody and the amount of the ALT2 polypeptide bound to thesecond antibody; wherein when the amount of the bound ALT2 polypeptideis sufficiently higher than the amount of the bound ALT1 polypeptide, itindicates that the animal has a disease or injury affecting tissuecontaining ALT2. The sample of bodily fluids can comprise a fluidselected from a group comprising blood, serum, lymph, urine, sweat,mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovialfluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleuralfluid. The tissue can be selected from a group comprising liver, brain,muscle, adipose tissue, and kidney.

Another embodiment of the invention is a method of diagnosing ordetecting injury or disease involving tissue which contains an ALTpolypeptide in an animal suspected of having the injury or disease. Themethod comprises: contacting a sample of bodily fluids from the animalsuspected of having the injury or disease with at least one firstantibody wherein the first antibody specifically binds to an ALTpolypeptide; detecting the first antibody which is bound to the ALTpolypeptide in the sample; and comparing the amount of the ALTpolypeptide in the sample of bodily fluids to an amount of ALT in thebodily fluids of an animal known not to have injury or disease involvingtissue which contains ALT polypeptide; wherein when the amount of ALT inthe bodily fluids of the sample is higher than the amount of ALTpolypeptide in the bodily fluids of the animal known not to have injuryor disease it indicates that the animal suspected has a disease orinjury affecting tissue containing ALT. The sample of bodily fluids cancomprise a fluid selected from a group including blood, serum, lymph,urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitialfluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginalfluid, and pleural fluid. The tissue can be selected from a groupcomprising liver, brain, muscle, adipose tissue, and kidney.

A further embodiment of the invention is a diagnostic kit for use indiagnosing damage or disease in tissue containing an ALT polypeptide ofthe present invention. The kit comprises a measurer for determining ameasurement of an ALT polypeptide in a sample of bodily fluids and anindicator for determining if the measurement falls in a range associatedwith damage or disease in the tissue containing the ALT of the presentinvention. In one embodiment, the ALT polypeptide is murine ALT2 of SEQID NO:2 or an amino acid sequence having about 95% homology thereto. Incertain embodiments, the homolog is an amino acid sequence of SEQ IDNO:5. In another embodiment, the ALT polypeptide is rat ALT2 of SEQ IDNO:5 or an amino acid sequence having about 95% homology thereto. Themeasurer can be selected from a group comprising a biologic assay, anantibody-based assay, an enzyme linked immunosorbent assay, a Westernblot, a rapid immunoassay, a radioimmunoassay, and combinations thereof.

Another embodiment of the invention is a diagnostic kit for use indiagnosing damage or disease in tissue containing an ALT polypeptide ofthe present invention. The kit comprises: an aliquot of antibodies thatbind specifically to an ALT polypeptide of the present invention;immunoassay reagents; and a control for determining if a measurement ofan ALT polypeptide of the present invention indicates a diagnosis ofdamage or disease in tissue containing an ALT polypeptide of the presentinvention. The control comprises instructions indicating that anincrease or decrease in the amount of the ALT polypeptide indicates adiagnosis for damage or disease in tissue containing the ALTpolypeptide. In a further embodiment, the kit further comprises analiquot of antibodies that bind specifically to ALT2 polypeptides of thepresent invention and a control for determining if a measurement of saidALT2 polypeptide indicates a diagnosis of damage or disease in tissuecontaining said ALT2 polypeptide. The control comprises instructionsindicating that an increase or decrease in the amount of ALT2 indicatesa diagnosis for damage or disease in tissue containing the ALTpolypeptide.

Another embodiment of the invention is a diagnostic kit for use in acondition associated with altered levels of an ALT polypeptide 1 inbodily fluids. The kit comprises a measurer for determining ameasurement of an ALT 1 polypeptide in a sample of bodily fluids and anindicator for determining if the measurement falls in a range associatedwith the condition.

Another embodiment of the invention is a diagnostic kit for use in acondition associated with altered levels of an ALT 2 in bodily fluids.The kit comprises a measurer for determining a measurement of an ALT 2polypeptide in a sample of bodily fluids and an indicator fordetermining if the measurement falls in a range associated with thecondition.

Another embodiment of the invention is a diagnostic kit for use in acondition associated with altered levels of at least one of an ALT 1 andan ALT2 in bodily fluids. The kit comprises: a measurer for determininga first measurement of an ALT 1 polypeptide in a sample of bodilyfluids; a measurer for determining a second measurement of ALT2polypeptide in the sample of bodily fluids; and an indicator fordetermining if the first and second measurements fall in a rangeassociated with the condition.

Still another embodiment of the invention is method of diagnosing acondition associated by altered levels of an ALT 1 in bodily fluids inan animal suspected of having the condition. The method comprises:contacting a sample of bodily fluids from the animal with at least oneantibody wherein the antibody specifically binds to an ALT 1polypeptide; detecting the antibody which is bound to the ALT 1polypeptide in the sample; and comparing the amount of the detectedantibody to a known quantity for an animal without the condition;wherein if the quantity of the detected antibody differs sufficientlyfrom the known quantity from an animal without the condition, indicatesthat the animal has the condition.

Another embodiment of the invention is a method of diagnosing acondition associated by altered levels of an ALT 2 in bodily fluids inan animal suspected of having the condition. The method comprises:contacting a sample of bodily fluids from the animal with at least oneantibody wherein the antibody specifically binds to an ALT 2polypeptide; detecting the antibody which is bound to the ALT 2polypeptide in the sample; and comparing the amount of the detectedantibody to a known quantity for an animal without the condition;wherein if the quantity of the detected antibody differs sufficientlyfrom the known quantity from an animal without the condition, indicatesthat the animal has the condition.

The sample of bodily fluids for the above methods and/or kits cancomprise a fluid selected from a group comprising of blood, serum,lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid,interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid,vaginal fluid, and pleural fluid.

The measurer for above methods and/or kits can be selected from thegroup comprising a biologic assay, an antibody-based assay, an enzymelinked immunosorbent assay, a Western blot, a rapid immunoassay, aradioimmunoassay, and combinations thereof.

Another embodiment of the invention is a method of producing an ALTpolypeptide. The method comprises: providing an ALT polynucleotidesequence in an expression vector; introducing the expression vector intoa host cell such that a recombinant host cell is produced; andsubjecting to the recombinant host cell to conditions such that the ALTpolypeptide is expressed. In one embodiment, the ALT polynucleotidesequence is selected from a group including SEQ ID NO:3 and SEQ ID NO:4.In another embodiment, the ALT polynucleotide sequence is selected froma group including SEQ ID NO:7 and SEQ ID NO:8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are a comparison of the sequence of murine ALT isoenzymes(mALT1 and mALT2) of this invention and the polypeptide sequence ofhuman ALT isoenzymes. FIG. 1A is a comparison between SEQ ID NO:1(murine ALT1) and human ALT1. FIG. 1B is a comparison between SEQ IDNO:2 (murine ALT2) and human ALT2. Peptide sequences are aligned usingBESFIT from the GCG package and amino acids are numbered to the right ofthe sequence. Identical amino acids are denoted by a vertical bar,strongly similar amino acids by a colon (:), and weakly similar aminoacids by a period (.).

FIG. 2 is Northern blots showing the expression of murine ALT mRNA.Blots from duplicate gels containing pooled total RNAs (15 μg/lane) from3 to 4 mice were probed separately with ³²P-labeled murine ALT1 or ALT2cDNAs. Hybridization signals were visualized by autoradiography. RNAloading from one of the duplicate gels is shown in the lower blot. Thesize of the mRNA transcripts is indicated in parentheses.

FIG. 3A is an electrophoresis gel where thirty microliters of E. coliextracts containing 100 to 150 μg of protein were analyzed on 4%-20%SDS-PAGE and stained with Coomassie Blue. Arrows indicate IPTG inducedprotein bands corresponding to mALT1 and mALT2.

FIG. 3B is a graph of ALT activity of soluble cell extracts of E. coliharboring plasmid pET28-mALT1 (ALT1), pET28-mALT2 (ALT2), or emptyvector pET28 (control) after IPTG induction.

FIGS. 4A and 4B are Northern blots of total RNA extracted from fattyliver of obese (ob/ob) and lean (+/?) mice blotted with ³²P-labeledmALT1 or mALT2 DNA probe. FIG. 4C is a graph of murine ALT expression.

FIG. 5 is a graph of ALT and AST activities in obese (ob/ob) and lean(+/?) mice measured for their ALT and AST activities. Activities areexpressed as mean±SD (n=7). *P<0.05 is considered statisticallysignificant.

FIG. 6 is a sequence alignment of ALT1 and ALT2 polypeptides from human,mouse (SEQ ID NO:1 and SEQ ID NO:2) and rat species (SEQ ID NO:6 and SEQID NO:5). Highly conserved amino acids (≧90%) are in capital letters andless conserved (<90% and ≧50%) are in small letters. Symbol “!” is forany amino acids of I or V, “$” for L or M, “%” for F or Y, and “#” isfor N, D, Q, E, B, or Z.

DEFINITIONS

Within the context of this specification, each term or phrase below willinclude the following meaning or meanings.

The terms “murine ALT polypeptide,” “murine ALT protein,” “murine ALT,”and “mALT” are interchangeable and generally refer to or include anyand/or all murine ALT polypeptides or isoenzymes, including murine ALT1,murine ALT2, and any variant, homolog, or fragment of murine ALT1 ormurine ALT2.

The terms “rat ALT polypeptide,” “rat ALT protein,” “rat ALT,” and“rALT” are interchangeable and generally refer to or include any and/orall rat (rattus) ALT polypeptides or isoenzymes, including rat ALT1, ratALT2, and any variant, homolog, or fragment of rat ALT1 or rat ALT2.

The terms “protein” and “polypeptide” are used interchangeably in bothsingular and plural forms, as are the terms “nucleic acid” and“polynucleotide.”

These and additional terms may be defined with additional language inthe remaining portions of the specification.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention covers the nucleotide and amino acid sequences ofmurine ALT, antibodies specific to murine ALT, and the use of thesepolypeptides, polynucleotides, and antibodies to diagnose variousdiseases and conditions in tissue that produce murine ALT, such as fattyliver, and to differentially diagnose liver injury caused by fatty liver(liver steatosis) and by alcohol, trauma, infection, toxicity, and othercauses of liver damage.

This invention also includes homologs and functional fragments of murineALT polypeptides as well as expression vectors containing murine ALTpolynucleotide sequences and recombinant host cells which contain anexpression vector containing murine ALT polynucleotide sequences.

This invention also includes homologs and functional fragments of ratALT polypeptides as well as expression vectors containing rat ALTpolynucleotide sequences and recombinant host cells which contain anexpression vector containing rat ALT polynucleotide sequences.

For this application, homology is often measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705 or the NCBI BLAST program). Suchsoftware matches similar sequences by assigning degrees of homology tovarious substitutions, deletions, substitutions, and othermodifications.

The term “functional fragments” include those fragments of SEQ ID NO:1and/or SEQ ID NO:2 and/or SEQ ID NO:5 and/or SEQ ID NO:6 and/or apolypeptide having about 95% sequence identity to that of the SEQ IDNO:1 and/or SEQ ID NO:2 and/or SEQ ID NO:5 and/or SEQ ID NO:6 and thatretains the function, activity, or immunobiological properties of saidALT polypeptide. One of skill in the art can screen for thefunctionality of a fragment by using the examples provided herein, wherefull length ALT1 and ALT2 are described. By “substantially identical” isalso meant an amino acid sequence which differs only by conservativeamino acid substitutions, for example, substitution of one amino acidfor another of the same class (e.g., valine for glycine, arginine forlysine, etc.) or by one or more non-conservative substitutions,deletions, or insertions located at positions of the amino acid sequencewhich do not destroy the function of the protein assayed (e.g., asdescribed herein). Preferably, such a sequence is at least 85%, and morepreferably from 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, to100% homologous at the amino acid level to SEQ ID NO:1 or SEQ ID NO:2 orSEQ ID NO:5 or SEQ ID NO:6.

Functional Equivalence

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the RNA sequence, according to thefollowing codon table:

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAG GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and messenger RNA sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the disclosed compositions, or corresponding DNA orRNA sequences which encode said peptides without appreciable loss oftheir biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within.+−.2 is preferred, those which are within .+−.1 are particularlypreferred, and those within .+−0.05 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−.1);glutamate (+3.0.+−.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within .+−.2 is preferred, those which are within .+−.1 areparticularly preferred, and those within .+−.0.5 are even moreparticularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

It will also be understood that amino acid sequences may includeadditional residues, such as additional N- or C-terminal amino acids andyet still be essentially as set forth in one of the sequences disclosedherein, so long as the sequence meets the criteria set forth above,including the maintenance of biological protein activity where proteinexpression is concerned. The addition of terminal sequences particularlyapplies to sequences, which may, for example, include variousunnaturally occurring amino acid sequences flanking either of the N- orC-termini to allow for facile covalent linkage to another molecule,i.e., a reporter molecule.

In non-limiting examples, the homolog may include a substitution in SEQID NO:1 of C56R, F109L, D126N, R133K, I152V, P158L, Q165R, A200S, R204H,A05T, A222T, D227G, R249H, C253G, R258H, V263A, E291K, G293R, M297L,A328S(*), V339T, E352A, M387L, T393A, S395T, K400A, R406K, E408A, R421S,Q434R, L439P, K440R, Q443E, D447E(*), C459R, Q477R, M491L, V496L(*),R502S(*), H503R, H510L or a combination thereof.

In a non-limiting example, the homolog may include a substitution in SEQID NO:6 of C56R, F109L, D126N, R133K, I152V, P158L, Q165R, S200A, R204H,A205T, A222T, D227G, R249H, C253G, R258H, V263A, K291E, G293R, M297L,S328A, V339T, E352A, M387L, T393A, S395T, K400A, R406K, E408A, R421S,Q434R, L439P, K440R, Q443E, E447D, C459R, Q477R, M491L, L496V, S502R,H503R, H510L or a combination thereof.

In a non-limiting example, the homolog may include a substitution in SEQID NO:5 of H24Q, D45E, M77L, H99Q, N123D, L1961, 1228V, D245N, L251V,R252Q, Q253E, Q321E, P326H, V392E, S407F, Q430H, L445F, S456A, K458Q,E501D, H508Q, L515I, K516N, K520Q, S522A or a combination thereof.

In a non-limiting example, the homolog may include a substitution in SEQID NO:2 of H24Q, D45E, M77L, H99Q, N123D, L196I, V2281, D245N, L251V,R252Q, Q253E, Q321E, H326P, E392V, F407S, Q430H, L445F, S456A, K458Q,D501E, H508Q, L515I, K516N, Q520K, S522A or a combination thereof.

By a “substantially pure polypeptide” is meant a ALT polypeptide thathas been separated from components that naturally accompany it.Typically, the polypeptide is substantially pure when it is at least60%, by weight, free from the proteins and other naturally occurringmolecules with which it is typically associated. Preferably, thepreparation is at least 75%, 80%, 90%, 95%, and most preferably at least99%, by weight, an ALT polypeptide. A substantially pure ALT polypeptidecan be obtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding the desired ALTpolypeptide; or by chemically synthesizing the protein. Purity can bemeasured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or by HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants that accompany it in its naturalstate. Thus, a protein that is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates is substantially free from its naturally associatedcomponents. Accordingly, substantially pure polypeptides include thosederived from eukaryotic organisms but synthesized in E. coli or otherprokaryotes. As will be appreciated by one skilled in the art followingthe teachings herein provided, the polynucleotide molecules of thepresent disclosure can be expressed in a variety of prokaryotic andeukaryotic cells using regulatory sequences, vectors, and methods wellestablished in the literature.

An ALT polypeptide produced according to the present description can bepurified using a number of established methods such as affinitychromatography using anti-mALT antibodies coupled to a solid support.Fusion proteins of an antigenic tag and an ALT polypeptide can bepurified using antibodies to the tag. Optionally, additionalpurification is achieved using conventional purification means such asliquid chromatography, gradient centrifugation, and gel electrophoresis,among others. Methods of protein purification are known in the art andcan be applied to the purification of recombinant ALT polypeptidedescribed herein.

Construction of ALT encoded fusion proteins is also contemplated. Fusionproteins typically contain additions, substitutions, or replacements ofone or more contiguous amino acids of the native ALT polypeptide withamino acid(s) from a suitable fusion protein partner. Such fusionproteins are obtained using recombinant DNA techniques generally wellknown by one of skill in the art. Briefly, DNA molecules encoding thehybrid ALT protein of interest are prepared using generally availablemethods such as PCR mutagenesis, site directed mutagenesis, and/orrestriction digestion and ligation. The hybrid DNA is then inserted intoexpression vectors and introduced into suitable host cells.

Recombinant gene expression vectors comprising a nucleic acid encodingan ALT protein of interest, or portions thereof, can be constructed in avariety of forms well-known in the art. Preferred expression vectorsinclude plasmids and cosmids. Expression vectors include one or morefragments of murine ALT. In one embodiment of this invention, anexpression vector comprises a nucleic acid encoding an ALTpolynucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:5 orSEQ ID NO:6. As used herein, the phrase “operatively encode” refers toone or more protein coding regions associated with those regulatorysequences required for expression of the polypeptide encoded by thecoding region. Examples of such regulatory regions including promoterbinding sites, enhancer elements, ribosome binding sites, and the like.Those of ordinary skill in the art following the teachings hereinprovided will be able to select regulatory sequences and incorporatethem into the recombinant expression vectors described herein withoutundue experimentation. For example, suitable regulatory sequences foruse in various eukaryotic and prokaryotic systems are described inAusubel, et al., Short Protocols in Molecular Biology, 3rd ed., JohnWiley & Sons, Inc, New York, 1997, which is hereby incorporated byreference in its entirety.

Expression vectors for use with ALT typically contain regulatorysequences derived from a compatible species for expression in thedesired host cell. For example, when E. coli is the host cell, the hostcell population can be typically transformed using pBR322, a plasmidderived from an E. coli species. (Bolivar, et al., Gene, 2: 95, 1977).pBR322 contains genes for ampicillin (AMPR) and tetracycline resistanceand thus provides easy means for identifying transformed cells.

Promoters suitable for use with prokaryotic hosts illustratively includethe betalactamase and lactose promoter systems (Chang, et al., Nature,275: 615, 1978; and Goeddel, et al., Nature, 281: 544, 1979), alkalinephosphatase, the tryptophan (trp) promoter system (Goeddel, NucleicAcids Res., 8: 4057, 1980) and hybrid promoters such as the taq promoter(de Boer, et al., Proc. Natl. Acad. Sci. USA, 80: 21-25, 1983). Otherfunctional bacterial promoters are also suitable. Their nucleotidesequences are generally known in the art, thereby enabling a skilledworker to ligate them to a polynucleotide which encodes the peptide ofinterest (Siebenlist, et al., Cell, 20: 269, 1980) using linkers oradapters to supply any required restriction sites.

In addition to prokaryotes, eukaryotic microbes such as yeast culturescan also be used as source for the regulatory sequences. Saccharomycescerevisiae is a commonly used eukaryotic host microorganism. Suitablepromoting sequences for use with yeast hosts include the promoters for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem., 255: 2073,1980) or other glycolytic enzymes (Hess, et al. J. Adv. Enzyme Reg. 7:149, 1968; and Holland, Biochemistry, 17: 4900, 1978) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degraded enzymes associated with nitrogen metabolism,metallothionine, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Yeast enhancers alsoare advantageously used with yeast promoters.

In another embodiment, a recombinant virus is used as the expressionvector. Exemplary viruses include the adenoviruses, adeno-associatedviruses, herpes viruses, vaccinia, or an RNA virus such as a retrovirusor an alphavirus. Preferably, the retroviral vector is a derivative of amurine or avian retrovirus. Preferably the alphavirus vector is derivedfrom Sindbis or Semliki Forest Virus. All of these vectors can transferor incorporate a gene for a selectable marker so that transduced cellscan be identified and generated.

By inserting one or more sequences of interest into the viral vector,along with another gene which encodes the ligand for a receptor on aspecific target cell, for example, the vector is now target specific.Retroviral vectors can be made target specific by inserting, forexample, a polynucleotide encoding a sugar, a glycolipid, or a protein.Preferred targeting is accomplished by using an antibody to target theretroviral vector, such as to the vicinity of a mucosal inductor site,using, for example, a mALT-specific antibody. Those of skill in the artknow of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the polynucleotides of interest.

Construction of suitable vectors containing desired coding, non-codingand control sequences employ standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to construct the plasmids required.

For example, for analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures can be used to transform a host celland successful transformants selected by antibiotic resistance whereappropriate. Plasmids from the transformants are prepared, analyzed byrestriction and/or sequenced by, for example, the method of Messing, etal., (Nucleic Acids Res., 9: 309, 1981), the method of Maxam, et al.,(Methods in Enzymology, 65: 499, 1980), or other suitable methods whichare known to those skilled in the art. Size separation of cleavedfragments is performed using conventional gel electrophoresis asdescribed, for example, by Maniatis, et al., (Molecular Cloning, pp.133-134, 1982).

Host cells can be transformed with the expression vectors describedherein and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisanfollowing the teachings herein provided.

In cloning murine ALT1 and murine ALT2, peptide sequences of human ALT1and ALT2 were used as probes to search the mouse murine expressedsequence tag (EST) database using tBLASTn. A search of the mouse ESTdatabase using human ALT1 and human ALT2 peptide sequences as probesyielded several highly homologous EST clones. Of them, IMAGE clones4195300 and 5065322 were fully sequenced and revealed the highesthomology to human ALT1 and ALT2, respectively, in the entireprotein-coding region. The DNA nucleotide sequences of these two cloneswere confirmed by sequencing analysis and are predicted to encodeproteins of 496 (clone 4195300) and 522 (clone 5065322) amino acids. Asshown in FIG. 1, comparison of the mouse and human peptide sequencesrevealed that IMAGE clone 4195300 shares about 87% identity and about89% similarity to human ALT1, but about 70% identity and about 72%similarity to human ALT2, whereas clone 5065322 shares about 93%identity and about 95% similarity with human ALT2, but about 69%identity to human ALT1. The cDNA clone 4195300 and the clone 5065322were thus determined to be murine ALT1 and murine ALT2, respectively.Sixty-seven percent of amino acids are identical in murine ALT1 andmurine ALT2; a similar degree of conservation, about 68%, is foundbetween human ALT1 and ALT2.

Cloning of rat ALT1 and ALT2 was achieved through bioinformatics byinterrogating the closest homolog in GenBank with murine and human ALT1and ALT2 protein sequences. The rat IMAGE clone 7113147 encodes aprotein that shares 95% and 98% identity to human and murine ALT2,respectively, but 68% and 67% identity to human and murine ALT1,respectively. Thus, the cDNA clone 7113147 was determined as rat ALT2.Rat ALT1 cDNA was cloned from rat liver first-strand cDNA by PCRamplification with high-fidelity DNA polymerase using primers based ratexpressed sequence tags (ESTs) which shares highest identity to murineand human ALT1 sequences. The resultant PCR fragment was cloned intoTOPO cloning vector (Invitrogen) and sequenced in full. The translatedprotein sequence shares high identities of 88% and 97% to human andmurine ALT1, respectively, but low identities of 70% and 68% to humanand murine ALT2, respectively. Thus, this clone was determined as ratALT1.

The gene expression of murine ALT1 and murine ALT2 was examined in mousetissues by Northern analysis. Male obese mice (ob/ob), littermatecontrol (+/?), and C57BL/6J, 6 to 8 weeks old, were obtained fromJackson Laboratory and euthanized with CO₂ according to protocolapproved by Institutional Animal Care and Use Committee. Tissues wereimmediately frozen in liquid nitrogen until use for RNA extraction orenzyme activity assay. Total RNA was prepared with Trizol, availablefrom Life Technologies Inc., Gaithersburg, Md., from the snap-frozentissues. For the tissue distribution study, pooled 15 μg of total RNAfrom 3 to 4 mice were electrophoresed on a 1.2% agarose gel and blottedto a Nitro-plus membrane, available from Schleicher & Schuell, Dassel,Germany. The DNA probes of murine ALT1 (1.4 kb) and murine ALT2 (2.4 kb)were derived from restriction enzyme digestion of IMAGE clone 4195300(Sal I/Not I) and clone 5065322 (Sal I/Not I), respectively. Probes wererandom-labeled with ³²P-dCTP, hybridization was carried out at 65° C. inRapid-hyb buffer, available from Amersham Biosciences, Piscataway, N.J.,and blots were washed twice with 0.5×SSC/1% SDS at 65° C. (stringentwash) and visualized by PhosphorImager, available from AmershamBiosciences, or x-ray film.

As shown in FIG. 2, the ≈3.3 kb murine ALT2 messenger RNA (mRNA) wasexpressed at high levels in muscle, liver, and white adipose tissue(WAT), at moderate levels in brain and kidney, and at a low level inheart. By contrast, the ≈1.8 kb murine ALT1 mRNA was highly expressed inliver and considerably in WAT, intestine, and colon tissue. As shown inFIG. 2 particular tissues selectively express one ALT isoenzyme over theother. For instance, murine ALT2 was significantly expressed, and murineALT1 barely expressed, in muscle and brain tissue. In contrast, boweltissue generally expressed only murine ALT1, and not murine ALT2.

To determine the chromosomal localization of DNA encoding murine ALT1and murine ALT2 (gpt1 and gpt2), the corresponding cDNAs were searchedagainst the mouse genome and localized gpt1 to murine chromosome 15D3and gpt2 to chromosome 8C2. Both of these regions are syntenic to thechromosomal regions where human gpt1 (chromosome 8q24.3) and gpt2(chromosome 16q12.2) reside. Full-length cDNAs of mALT1 (BC022625) andmALT2 (BC34219) were searched against the mouse genome sequence databasewith BLASTn, and their chromosomal localizations were determined byMapViewer.

The coding region of mALT1 cDNA was amplified by polymerase chainreaction (PCR) at 28 cycles at 94° C. for 30 seconds, 56° C. for 30seconds, and 72° C. for 1.5 minutes, with a final extension of 7 minutesat 72° C. using the Turbo Pfu PCR system (Stratagene) with anNdeI-linked primer, p1408 5′-GGAAGATCTCATATGGCCTCACAAAGGAATGAC-3′(nt<106-126, BC022625; SEQ ID NO:9), and a NotI;-linked primer, p14095′-AATGCGGCCGCTCAGGAGTACTCATGAGTGAA-3′ (1596-1576, BC022625; SEQ IDNO:10), using IMAGE clone 4195300 as a template. The resulting PCRproduct was digested with NdeI/NotI and subcloned into pET28a, availablefrom Novagen, Madison, Wis., creating plasmid pET28-mALT1. The absenceof mutations in the inserted murine ALT1 cDNA was verified by DNAsequence analysis. The same approach was used to clone the coding regionof murine ALT2 cDNA from IMAGE clone 5065322 into pET28a by PCR usingIMAGE clone 5065322 as template with primer p14055′-GGAAGATCTCCATGGCCCATATGCAGCGGGCAGCGGTGCTGGT-3′ (nt 128-150, BC034219;SEQ ID NO:11) and p1406 5′-AATGCGGCCGCTCATGAGTACTGCTCCAGGAA-3′ (nt1696-1676, BC034219; SEQ ID NO:12), creating plasmid pET28-mALT2.

To express mALT1 and mALT2 proteins, plasmid pET28-mALT1, pET28-mALT2,or empty vector pET28 were used to transform competent E. coli. (TunerDE3, available from Novagen). A fresh colony of the transformants wasgrown in 50 ml LB media containing 30 μg/ml kanamycin to an OD₆₀₀ of0.7, at which time isopropyl-beta-D-thiogalactopyranoside (IPTG) wasadded (1 mmol, final concentration) to induce expression of therecombinant proteins. Cell pellets were harvested from 20 ml culturesbefore and after 4 hours of induction and were resuspended in 5 ml of TEbuffer (10 mmol Tris-HCl (pH 7.4), 0.1 mmol ethylenediaminetetraaceticacid), followed by a brief sonication, 3×10 seconds, setting 3 using aFisher 550 Sonic Dismembrator. Cell lysates were centrifuged at 10,000 gfor 15 minutes at 4° C., and supernatants were analyzed immediately forenzyme activity and protein analysis.

Soluble fractions of cell lysates were assayed for ALT activity andsubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis. As shown in FIG. 3A, under induction of IPTG,significant ALT activity was observed in cell lysates from bacteriatransformed with pET28-mALT1 (2.62 units/mg protein) and pET28-mALT2(0.72 units/mg protein), compared to empty vector control (0.19 units/mgprotein). Accordingly, as shown in FIG. 3B, protein bands at approximatemolecular weights (MW) of 58 kd and 62 kd were clearly visible inbacterial cell pellets after IPTG induction, corresponding to murineALT1 (calculated MW=55 kd) and murine ALT2 (calculated MW=58 kd). Itshould be noted that murine ALT activities detected in the above celllysates do not reflect the specific activity of the murine ALTisoenzymes, as most of the recombinant proteins were expressed ininsoluble fraction of inclusion body, and, therefore, the actual amountof ALT in the cell lysates was not known. Nevertheless, these dataconfirm that murine ALT1 and ALT2 cDNAs encode functional murine ALT.

The ALT activity of the bacterially expressed recombinant murine ALTproteins was confirmed using a GPT Optimized Alanine Aminotransferasekit, available from Sigma Diagnostics (catalog no. DG159-K), St. Louis,Mo., according to manufacturer instructions. Briefly, 0.5 ml of celllysate was incubated with a 2.5 ml mixture of reagent A and B containingL-alanine, nicotinamide adenine dinucleotide, Lactate Dehydrogenase, and2-oxoglutarate at 25° C. Absorbance at 340 nm was recorded at 1, 2, and3 minutes after incubation. The slope of absorbance decrease isproportional to ALT activity. Protein concentration of cell lysates weredetermined by Coomassie Brilliant Blue G250 (BioRad) using bovine serumalbumin as a standard. Final ALT activities were corrected by proteinconcentration of cell lysates. One unit of ALT activity was defined asthe amount of enzyme that catalyzes the formation of 1 μmol/liter ofnicotinamide adenine dinucleotide per minute at 25° C.

To demonstrate hepatic ALT and aspartate aminotransferase (AST)activity, a piece of snap-frozen liver (50-60 mg) was thawed on ice andthen minced and homogenized in 19 volumes of TE buffer (wt/vol) withDounce homogenizer (40 times). The resulting homogenate was furthersonicated (3×10 seconds, setting 4, using a Fisher 550 SonicDismembrator) followed by centrifugation at 10,000 rpm for 15 minutes at4° C. The supernatant was assayed for hepatic ALT using an L-type GPT J2kit, available from Wako Chemicals, Osaka, Japan, according to themanufacturer's instructions. AST activity was measured with an AST/GOTLiqui-UV kit, available from Stanbio Laboratories, Boerne, Tex.,according to the manufacturer's instructions.

Murine ALT2 gene expression is specifically induced in fatty liver. Thedistinctive tissue distribution patterns of murine ALT1 and ALT2 mRNAsare likely due to a difference in their gene regulation. Gene expressiondifferences were examined in fatty livers of obese mice (ob/ob). FIGS.4A-C demonstrate the increased mALT2 gene expression in fatty liver.FIGS. 4A and 4B are duplicate blots containing 15 μg of total RNAextracted from fatty liver of obese (ob/ob) and lean (+/?) mice blottedwith ³²P labeled mALT1 or mALT2 DNA probe. Hybridization signals werevisualized and quantitated by PhosphorImager. In FIGS. 4A and 4B, RNAloading from one of the duplicate gels is shown in lower blot. FIG. 4Cshows data expressed as mean±SD (n=3). *P<0.05 is consideredstatistically significant. As shown in FIGS. 4A-C, compared to thenormal liver of the lean mice control (+/?), the expression of murineALT2 mRNA was elevated about 1-fold, but that of murine ALT1 remainedunchanged. Furthermore, a significant elevation (30%, P<0.01) of murineALT enzymatic activity was observed in fatty liver relative to normalliver. The data indicates that murine ALT2 induction is most generallyresponsible for the increased murine ALT activity in fatty liver.Interestingly, as shown in FIG. 5, AST activity was barely increased infatty liver (5%, P=0.5) compared with normal liver.

Thus the invention provides molecular cloning of animal homologues ofhuman ALT1 and ALT2. ALT is an important intermediary enzyme involved inthe metabolism of amino acids, glucose, and possibly fatty acids and iswell known for its use as a surrogate marker for liver damage inclinical diagnostics. In addition to the dissimilarity of the peptidesequences of murine ALT1 and murine ALT2, the mouse genes reside onseparate chromosomes and have distinct tissue distributions and possiblycellular localizations. The murine ALT isoenzymes behave discordantly invarious clinical conditions. In other words, under certain clinicalconditions, one isoenzyme may be elevated but not the other, or viceversa. By virtue of this feature, individual murine ALT isoenzymes canbe better diagnostic markers than a total murine ALT activity.Similarly, individual rat ALT isoenzymes can be better diagnosticmarkers than a total rat ALT activity.

ALT activities are present in many tissues, including liver, heart,kidney, muscle, brain, and adipose tissue in rodents. Northern blot dataindicate one or both of the murine ALT genes are expressed in thetissues where ALT activity has been observed. Murine ALT1 is mainlyexpressed in liver and bowels, whereas murine ALT2 is highly expressedin muscle, liver, fat, and kidney, a tissue pattern reminiscent of humanALT1 and ALT2 tissue distribution. The conservation among rat, murineand human ALT isoenzymes in protein sequence, gene localization, andtissue distribution forms a basis for using the rat and/or mouse as amodel for exploring the diagnostic value of ALT isoenzymes.

ALT and AST activity levels have been used in clinic diagnostics formany years. Elevation of these two enzyme activities in serum areregarded as evidence of liver damage, as in viral hepatitis, NASH, ordrug hepatotoxicity. However, the mechanism for the serum ALT increasehas not been well understood and has been thought to be caused by“leakage” of the cellular enzyme into the systemic circulation.Moreover, which ALT isoenzyme is responsible for the serum elevation hasnot been known because the current ALT assay measures total catalyticactivity of ALT, presumably the combined activity of ALT1 and ALT2.

Molecular cloning of ALT isoenzymes in rat and mice provides a means tostudy the underlying mechanism(s) as well as an interpretation ofclinical ALT observations. For example, ALT elevation in muscle diseasemay be due to a “leak” of ALT, presumably ALT2, from muscle, where ALT2,but not ALT1, is abundantly expressed. Similarly, a specific ALTisoenzyme may be induced in a given clinical condition.

The hepatic murine ALT1 and murine ALT2 gene expression were examined inobese mice because hepatic steatosis is associated with this geneticallyobese model. Indeed, murine ALT2, but not murine ALT1, gene expressionis specifically induced. Furthermore, total murine ALT enzymaticactivity is increased by 30% in fatty liver over nonfatty liver,suggesting that murine ALT2 may be primarily responsible for theincreased serum ALT activity in liver steatosis. Interestingly, ASTactivity remains unchanged in the same condition. These animal findingsare in agreement with clinical observations in which serum ALT isgenerally increased to a greater extent than AST in patients with liversteatosis.

Additionally, the ALT isoenzymes may be present in different cellularcompartments, which can also be utilized for diagnostic purposes: therelease of a given ALT isoenzyme into circulation reflects the nature ofthe liver damage. It has been shown that serum mitochondrial AST contentis specifically increased in patients treated with halothane, andsuggested that this AST isoenzyme is a sensitive marker forhalothane-induced hepatic injury. Both cytosolic and mitochondrialmurine and rat ALT activities were found in liver, kidney, and skeletaland cardiac muscles. At present, which ALT isoenzyme is cytosolic ormitochondrial is not certainly clear. ALT isoenzyme specific antibodieshelp to elucidate the cellular localization of ALT isoenzymes and theirchanges in disease states.

In certain embodiments of the present invention, ALT isoform-specificantibody is required for establishment of isoform-specific detection,such as by enzyme-linked immunoabsorbant assay (ELISA). For thispurpose, a recombinant ALT1 polypeptide and/or a recombinant ALT2polypeptide is generated in bacteria and purified to homogeniety. About2 mg of the purified protein is used to immunize mice for generatingantibody specific for murine ALT or, alternatively, to immunize rats forgenerating antibody specific for rat ALT. As a result, a monoclonalhybridoma against a isoform of an ALT polypeptide of the presentinvention is determined by determining binding specificity to said ALTpolypeptide. One such method is Western blot analysis in which about 20ng of ALT2, of ALT1, and of bovine serum albumin are load onto anSDS-PAGE gel, electrophoresised, and blotted to PVDF membrane, which isincubated with cell culture media of the indicated hybridoma cells(1:100 dilution) and visualized by chemiluminescence. Cross-reaction ofthe antibody produced by the hyridoma cell identified above to the otherisoform is also determined. The identified isoform-specific antibody isemployed in the methods of the present invention, such as an ALTisoform-specific ELISA.

Differences in ALT isoenzyme in tissue distribution, gene regulation,and possible cellular localization suggest that the ability to measureALT isoenzyme-specific activity levels is a significant improvement overmeasurement of total ALT activity in clinical diagnostics. The cloningof murine and rattus homologues of human ALT isoenzymes provides a noveltool for clinical applications of ALT isoenzymes as molecular markersfor nonalcoholic fatty liver diseases as well as other clinicalconditions.

Data herein are generally presented as mean±standard deviation (SD).Statistical significance was determined by Student unpaired t test. Pvalue less than 0.05 was considered significant.

While the embodiments of the invention described herein are presentlypreferred, various modifications and improvements can be made withoutdeparting from the spirit and scope of the invention. The scope of theinvention is indicated by the appended claims, and all changes that fallwithin the meaning and range of equivalents are intended to be embracedtherein.

1. An isolated and purified alanine transaminase (ALT) polypeptide,wherein the polypeptide is selected from a polypeptide comprising theamino acid sequence of SEQ ID NO: 2 and a polypeptide sequencecomprising at least 98% identity to the amino acid sequence of SEQ IDNO:
 1. 2. An isolated and purified alanine transaminase (ALT)polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 5.3. A method of producing the ALT polypeptide of claim 1, comprising:providing an ALT polynucleotide sequence for the ALT polypeptide ofclaim 1 in an expression vector; introducing the expression vector intoa host cell such that a recombinant host cell is produced; andsubjecting to the recombinant host cell to conditions such that the ALTpolypeptide of claim 1 is expressed.
 4. The isolated and purifiedalanine transaminase (ALT) polypeptide of claim 1, wherein thepolypeptide sequence comprises at least 99% identity to the amino acidsequence of SEQ ID NO:
 1. 5. The isolated and purified alaninetransaminase (ALT) polypeptide of claim 1, wherein the polypeptidecomprises the sequence of SEQ ID NO: 1.